Abstract
Antisense strategy is an attractive substitute for knockout mutations created for gene silencing. mce genes have been shown to be involved in mycobacterial uptake and intracellular survival. Here we report reduced expression of mce4A and mce1A genes of Mycobacterium tuberculosis using antisense technology. For this, 1.1. kb region of mce4A and mce1A was cloned in reverse orientation in pSD5 shuttle vector, resulting into antisense constructs pSD5-4AS and pSD5-1AS, respectively. In M. tuberculosis H37Rv approximately 60% reduction in Mce4A and 66% reduction in expression of Mce1A protein were observed. We also observed significantly reduced intracellular survival ability of both antisense strains in comparison to M. tuberculosis containing pSD5 alone. RT-PCR analysis showed antisense did not alter the transcription of upstream and downstream of mceA genes of the respective operon. The colony morphology, in vitro growth characteristics and drug susceptibility profile of the antisense construct remained unchanged. These results demonstrate that antisense can be a promising approach to assign function of a gene in a multiunit operon and could be suitably applied as a strategy.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 780-787 |
| Number of pages | 8 |
| Journal | Microbiological Research |
| Volume | 169 |
| Issue number | 9-10 |
| DOIs | |
| State | Published - 2014 |
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Keywords
- Antisense
- Mce
- Mycobacterium tuberculosis
- Polycistronic operon
ASJC Scopus subject areas
- Microbiology
- Medicine(all)
Cite this
Functional analysis of mce4A gene of Mycobacterium tuberculosis H37Rv using antisense approach. / Chandolia, Amita; Rathor, Nisha; Sharma, Monika; Saini, Neeraj K.; Sinha, Rajesh; Malhotra, Pawan; Brahmachari, Vani; Bose, Mridula.
In: Microbiological Research, Vol. 169, No. 9-10, 2014, p. 780-787.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Functional analysis of mce4A gene of Mycobacterium tuberculosis H37Rv using antisense approach
AU - Chandolia, Amita
AU - Rathor, Nisha
AU - Sharma, Monika
AU - Saini, Neeraj K.
AU - Sinha, Rajesh
AU - Malhotra, Pawan
AU - Brahmachari, Vani
AU - Bose, Mridula
PY - 2014
Y1 - 2014
N2 - Antisense strategy is an attractive substitute for knockout mutations created for gene silencing. mce genes have been shown to be involved in mycobacterial uptake and intracellular survival. Here we report reduced expression of mce4A and mce1A genes of Mycobacterium tuberculosis using antisense technology. For this, 1.1. kb region of mce4A and mce1A was cloned in reverse orientation in pSD5 shuttle vector, resulting into antisense constructs pSD5-4AS and pSD5-1AS, respectively. In M. tuberculosis H37Rv approximately 60% reduction in Mce4A and 66% reduction in expression of Mce1A protein were observed. We also observed significantly reduced intracellular survival ability of both antisense strains in comparison to M. tuberculosis containing pSD5 alone. RT-PCR analysis showed antisense did not alter the transcription of upstream and downstream of mceA genes of the respective operon. The colony morphology, in vitro growth characteristics and drug susceptibility profile of the antisense construct remained unchanged. These results demonstrate that antisense can be a promising approach to assign function of a gene in a multiunit operon and could be suitably applied as a strategy.
AB - Antisense strategy is an attractive substitute for knockout mutations created for gene silencing. mce genes have been shown to be involved in mycobacterial uptake and intracellular survival. Here we report reduced expression of mce4A and mce1A genes of Mycobacterium tuberculosis using antisense technology. For this, 1.1. kb region of mce4A and mce1A was cloned in reverse orientation in pSD5 shuttle vector, resulting into antisense constructs pSD5-4AS and pSD5-1AS, respectively. In M. tuberculosis H37Rv approximately 60% reduction in Mce4A and 66% reduction in expression of Mce1A protein were observed. We also observed significantly reduced intracellular survival ability of both antisense strains in comparison to M. tuberculosis containing pSD5 alone. RT-PCR analysis showed antisense did not alter the transcription of upstream and downstream of mceA genes of the respective operon. The colony morphology, in vitro growth characteristics and drug susceptibility profile of the antisense construct remained unchanged. These results demonstrate that antisense can be a promising approach to assign function of a gene in a multiunit operon and could be suitably applied as a strategy.
KW - Antisense
KW - Mce
KW - Mycobacterium tuberculosis
KW - Polycistronic operon
UR - http://www.scopus.com/inward/record.url?scp=84904751221&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84904751221&partnerID=8YFLogxK
U2 - 10.1016/j.micres.2013.12.008
DO - 10.1016/j.micres.2013.12.008
M3 - Article
C2 - 24556072
AN - SCOPUS:84904751221
VL - 169
SP - 780
EP - 787
JO - Microbiological Research
JF - Microbiological Research
SN - 0944-5013
IS - 9-10
ER -